1. Using the genome
Studying expression of all genes simultaneously
Microarrays: “reverse Northerns”
High-throughput sequencing
“Re-sequencing” to detect variation
Sequencing all mRNA to quantitate gene expression
Sequencing all mRNA to identify and quantitate splicing variants
Sequencing all RNA to identify and quantitate ncRNA
Surprises
Over half of human genome is transcribed even though only 1% is protein-coding sequence

2. Surprises
Over half of human genome is transcribed even though only 1% is protein-coding sequence
98% of RNA made is non-coding

3. Surprises
Over half of human genome is transcribed even though only 1% is protein-coding sequence
98% of RNA made is non-coding
Fraction increases with organism’s complexity

4. Surprises
Over half of human genome is transcribed even though only 1% is protein-coding sequence
98% of RNA made is non-coding
Fraction increases with organism’s complexity
Incredible diversity of functions!

5. ncRNA
Structural
rRNA
tRNA
snRNA
snoRNA
cleavage: Rnases P & MRP, U3, snR30, etc
Regulatory
Small
siRNA
miRNA
Long
Activator
Enhancer
silencing

6. Surprises
Over half of human genome is transcribed even though only 1% is protein-coding sequence
At least 10% of human genes have anti-sense copies

7. At least 10% of human genes have anti-sense copies
Surprises
Over half of human genome is transcribed even though only 1% is protein-coding sequence
At least 10% of human genes have anti-sense copies
BACE-1 AS may contribute to Alzheimer’s by increasing production of Aβ
BACE 1 beta-site amyloid precursor protein cleaving enzyme 1 is an aspartic-acid protease important in the formation of myelin sheaths
First step in forming A-beta implicated in Alzheimers (gamma-secretase finishes the job of forming the 40 aa peptide)

8. Surprises
Over half of human genome is transcribed even though only 1% is protein-coding sequence
At least 10% of human genes have anti-sense copies
Nearly every gene that can be is alternatively spliced in some tissue or condition.
~ 400 genes were spliced differently in male
& female human brains

9. Surprises
Over half of human genome is transcribed even though only 1% is protein-coding sequence
At least 10% of human genes have anti-sense copies
Nearly every gene that can be alternatively spliced is alternatively spliced in some tissue or condition.
Many genes have RNA polymerase II paused about 100 bp from the start

10. Using the genome
Studying expression of all genes simultaneously
Epigenetics: modifications to DNA that alter gene expression
Bisulfite sequencing to detect C methylation

11. Using the genome
Bisulfite sequencing to detect C methylation

12. Using the genome
Bisulfite sequencing to detect C methylation
ChIP-chip or ChIP-seq to detect chromatin modifications
17 mods are associated with active genes in CD-4 T cells

13. Using the genome
Epigenetics: various chromatin modifications are associated with activated & repressed genes
Acetylation, egH3K9Ac, is associated with active genes

14. Using the Genome
various chromatin modifications are associated with activated & repressed genes
Acetylation, egH3K9Ac, is associated with active genes
Phosphorylation of H2aS1, H2aT119, H3T3, H3S10 & H3S28 shows condensation

15. Using the Genome
Phosphorylation of H2aS1, H2aT119, H3T3, H3S10 & H3S28 shows condensation
Phosphorylation of Histone H3 correlates with activation of heat shock genes!

16. Using the Genome
Acetylation, egH3K9Ac, is associated with active genes
Phosphorylation = condensation
Ubiquitination of H2A and H2B (mostly) = repression
Methylation is more complex
H3K36me3 = on
H3K27me3 = off

17. Using the Genome
Methylation is more complex:
H3K36me3 = on
H3K27me3 = off
H3K4me1 = off
H3K4me2 = primed
H3K4me3 = on

18. Histone code
Modifications tend to group together: genes with H3K4me3 also have H3K9ac

19. Histone code
Modifications tend to group together: genes with H3K4me3 also have H3K9ac
Cytosine methylation is also associated with repressed genes

20. Generating the histone code
Histone acetyltransferases add acetic acid

21. Generating the histone code
Histone acetyltransferases add acetic acid
Many HAT proteins: mutants are very sick!

22. Generating the histone code
Histone acetyltransferases add acetic acid
Many HAT proteins: mutants are very sick!
HATs are part of many complexes

23. Generating the histone code
Bromodomains specifically bind acetylated lysines

24. Generating the histone code
Bromodomains specifically bind acetylated lysines
Found in transcriptional activators & general TFs

25. Generating the histone code
acetylated lysines
Deacetylases “reset” by removing the acetate

26. Generating the histone code
acetylated lysines
Deacetylases “reset” by removing the acetate
Deacetylase mutants are sick!

27. Generating the histone code
Deacetylases “reset” by removing the acetate
Deacetylase mutants are sick!
Many drugs are histone deacetylase inhibitors

28. Generating the histone code
Many drugs affect histone deacetylases
Resveratrol activates Sirtuin 1

29. Generating the histone code
Many drugs affect histone deacetylases
Resveratrol activates Sirtuin 1
Many drugs are histone deacetylase inhibitors
SAHA = suberanilohydroxamic acid = vorinostat
Merck calls it Zolinza, treats cutaneous T cell lymphoma

30. Generating the histone code
Many drugs affect histone deacetylases
Resveratrol activates Sirtuin 1
Many drugs are histone deacetylase inhibitors
SAHA = suberanilohydroxamic acid = vorinostat
Merck calls it Zolinza, treats cutaneous T cell lymphoma
Binds HDAC active site & chelates Zn2+

31. Generating the histone code
When coupled SAHA to
PIPS (pyrrole-imidazole
Polyamides) got gene-
specific DNA binding &
gene activation

32. Generating the histone code
CDK8 kinases histones to repress transcription

33. Generating the histone code
CDK8 kinases histones to repress transcription
Appears to interact with mediator to block transcription

34. Generating the histone code
CDK8 kinases histones to repress transcription
Appears to interact with mediator to block transcription
Phosphorylation of Histone H3 correlates with activation of heat shock genes!

35. Generating the histone code
CDK8 kinases histones to repress transcription
Appears to interact with mediator to block transcription
Phosphorylation of Histone H3 correlates with activation of heat shock genes!
Phosphatases reset the genes

36. Generating the histone code
Acetylation, egH3K9Ac, is associated with active genes
Phosphorylation shows condensation
Ubiquitination of H2A and H2B shows repression & marks DNA damage

37. Generating the histone code
Ubiquitination of H2A and H2B shows repression & marks DNA damage
Rad6 proteins ubiquitinate histone H2B to repress transcription

38. Generating the histone code
Rad6 proteins ubiquitinate histone H2B to repress transcription
Other proteins ubiquitinate H2B to enhance transcription!

39. Generating the histone code
Rad6 proteins ubiquitinate histone H2B to repress transcription: others enhance it!
Polycomb proteins ubiquitinate histone H2A to silence genes

40. Generating the histone code
Rad6 proteins ubiquitinate histone H2B to repress transcription: others enhance it!
Polycomb proteins ubiquitinate H2A to silence genes
Multiple de-ubiquitinases have been identified

41. Generating the histone code
Many proteins methylate histones: highly regulated!

42. Generating the histone code
Many proteins methylate histones: highly regulated!
Methylation status determines gene activity

43. Generating the histone code
Many proteins methylate histones: highly regulated!
Methylation status determines gene activity
Mutants (eg Curly leaf) are unhappy!

44. Generating the histone code
Many proteins methylate histones: highly regulated!
Methylation status determines gene activity
Mutants (eg Curly leaf) are unhappy!
Chromodomain protein HP1 can tell the difference between H3K9me2 (yellow) & H3K9me3 (red)

45. Generating the histone code
Chromodomain protein HP1 can tell the difference between H3K9me2 (yellow) & H3K9me3 (red)
Histone demethylases have been recently discovered

46. Generating methylated DNA
Si RNA are key: RNA Pol IV generates antisense or foldback RNA, often from TE

47. Generating methylated DNA
Si RNA are key: RNA Pol IV generates antisense or foldback RNA, often from TE
RDR2 makes it DS, 24 nt siRNA are generated by DCL3

48. Generating methylated DNA
RDR2 makes it DS, 24 nt siRNA are generated by DCL3
AGO4 binds siRNA, complex binds target & Pol V

49. Generating methylated DNA
RDR2 makes it DS, 24 nt siRNA are generated by DCL3
AGO4 binds siRNA, complex binds target & Pol V
Pol V makes intergenic RNA, associates with AGO4-siRNA to recruit “silencing Complex” to target site

50. Generating methylated DNA
RDR2 makes it DS, 24 nt siRNA are generated by DCL3
AGO4 binds siRNA, complex binds target & Pol V
Pol V makes intergenic RNA, associates with AGO4-siRNA to recruit “silencing Complex” to target site
Amplifies signal!
extends meth-
ylated region